Metabolically engineered lactic acid bacteria and their use

ABSTRACT

Mutants of lactic acid bacteria including  Lactococcus lactis  which are defective in pyruvate formate-lyase production and/or in their lactate dehydrogenase (Ldh) production and methods of isolating such mutants or variants are provided. The mutants are useful in the production of food products or in the manufacturing of compounds such as diacetyl, acetoin and acetaldehyde and as components of food starter cultures. In particular,  Lactococcus lactis  DN223 deposited under the accession No. DSM 11036.

The present application is a U.S. National stage of PCT applicationPCT/DK97/00335, filed Aug. 20, 1997, which claims priority from and is acontinuation-in-part of U.S. application No. 08/701,459, filed Aug. 22,1996, now abandoned, the entire contents of both of which being herebyincorporated herein by reference.

CROSS-REFERENCE TO RELATED APPLICATIONS

Arnau, et al., PCT/DK97/00336, is related to metabolically engineeredlactic acid bacteria and means for providing same. It is acontinuation-in-part of Ser. No. 08/701,458, filed Aug. 22, 1996.

FIELD OF INVENTION

The present invention relates to the field of lactic acid bacterialstarter cultures and in particular there is provided the means ofmetabolically engineering such bacteria to obtain mutants or variantshereof which, when they are used in the manufacturing of fermented foodproducts produce increased amounts of desirable metabolites or reducedamounts of less desirable metabolites.

TECHNICAL BACKGROUND AND PRIOR ART

Lactic acid bacteria are used extensively as starter cultures in thefood industry in the manufacture of fermented products including milkproducts such as e.g. yoghurt and cheese, meat products, bakeryproducts, wine and vegetable products. Lactococcus species includingLactococcus lactis are among the most commonly used lactic acid bacteriain dairy starter cultures. However, several other lactic acid bacteriasuch as Leuconostoc species, Pediococcus species, Lactobacillus speciesand Streptococcus species. Species of Bifidobacteri-um, a group ofstrict anaerobic bacteria, are also commonly used in food startercultures alone or in combination with lactic acid bacterial species.

When a lactic acid bacterial starter culture is added to milk or anyother food product starting material under appropriate conditions, thebacteria grow rapidly with concomitant conversion of citrate, lactose orother sugar compounds into lactic acid/lactate and possibly other acidsincluding acetate, resulting in a pH decrease. In addition, severalother metabolites are produced during the growth of lactic acidbacteria. These metabolites include ethanol, formate, acetaldehyde,α-acetolactate, acetoin, diacetyl, and 2,3 butylene glycol (butanediol).Among these metabolites, diacetyl is an essential flavour compound whichis formed during fermentation of the citrate-utilizing species of e.g.Lactococcus, Leuconostoc and Lactobacillus. Diacetyl is formed by anoxidative decarboxylation (FIG. 1) of α-acetolactate which is formed bythe action of α-acetolactate synthetase (Als) from two molecules ofpyruvate. Pyruvate is a key intermediate of several lactic acidbacterial metabolic pathways including the citrate metabolism and thedegradation of lactose or glucose to lactate. The pool of pyruvate inthe cells is critical for the flux through the metabolic pathway leadingto diacetyl, acetoin and 2,3 butylene glycol (butanediol) via theintermediate compound α-acetolactate due to the low affinity ofα-acetolactate synthetase for pyruvate.

Pyruvate is converted to formate and acetyl coenzyme A (acetyl CoA)(FIG. 1) by the action of pyruvate formate-lyase (Pfl). This conversiontakes place only under anaerobic conditions (Frey et al. 1994). Pfl isinactivated even at low levels of oxygen, and a switch from anaerobic toaerobic conditions will lead to significant changes in metabolic endproduct profiles in lactic acid bacteria with complete disappearance ofethanol and formate (Hugenholtz, 1993). Another factor which regulatesthe activity of Pfl is the pH. The pH optimum of Pfl is about 7(Hugenholtz, 1993).

An alternative pathway for the formation of acetyl CoA from pyruvate(FIG. 1) in a lactic acid bacterium is by the activity of the pyruvatedehydrogenase complex (PDC). In contrast to Pfl, PDC has a very lowactivity under anaerobic conditions due to the inhibitory effect of NADHon that enzyme (Snoep et al. 1992). This enzyme requires the presence oflipoic acid as a co-factor to be active.

Additionally, acetyl CoA can be produced in lactic acid bacteria fromacetate under aerobic as well as under anaerobic conditions.

Accordingly, it is conceivable that the pyruvate pool is increased underanaerobic conditions if the lactic acid bacterial strain is defective inenzyme systems involved in pyruvate consumption, including Pfl. Asmentioned above, an increased pyruvate pool may lead to an increasedflux from pyruvate towards acetoin and diacetyl or other metabolitesderived from α-acetolactate. Thus, it is to be expected that fermentedfood products which are produced by using a lactic acid bacterialstarter culture having a reduced Pfl activity or completely lacking suchactivity contain an increased amount of acetoin or other of the abovemetabolites. Conversely, the such starter cultures may produce reducedamounts of other metabolites, including ethanol and acetate andpossibly, acetaldehyde.

Recent studies have shown that when L. lactis is lacking the lactatedehydrogenase (Ldh) which is involved in the major pyruvate consumingpathway leading to lactate, more pyruvate is directed towards acetoinand butanediol via α-acetolactate, possibly resulting in increasedformation of the intermediate product diacetyl (Platteeuw et al., 1995;Gasson et al., 1996).

Overproduction of α-acetolactate synthetase in Lactococcus lactis asanother approach of metabolically engineering lactic acid bacteria toproduce increased amounts of diacetyl has been disclosed by Platteeuw etal. 1995.

The potential of using L. lactis strains with reduced pyruvateformate-lyase activity as a means of increasing diacetyl formation ismentioned by Hugenholtz, 1993. It is suggested by this author that thecombination of three strategies: 1) Ldh inactivation by mutation/geneticengineering, 2) Pfl inactivation by aeration and/or low pH and 3)acetolactate decarboxylase (ALD) inactivation by mutation/geneticengineering could result in a high production of α-acetolactate fromlactose.

However, the suggested inactivation of Pfl activity by aeration and/orlow pH is not feasible or possible in the industrial production oflactic acid bacterially fermented dairy products or other fermented foodproducts, as the production hereof generally takes place underessentially anaerobic conditions. Furthermore, the pH of the startingmaterials including milk is typically about 7 and it is generally notdesirable to lower the pH of the food material to be fermented.

Whereas it has been suggested to modify the Pfl activity of lactic acidbacteria as a means of changing their production of metabolites in adesirable direction by manipulating the growth conditions, there havebeen no suggestions in the prior art to utilize metabolically engineeredlactic acid bacteria which have a modified Pfl activity underindustrially appropriate and feasible culturing conditions.

A method that allows isolation of mutants of gram-negative bacteriadevoid of Pfl activity has been disclosed by Pascal et al., 1974. Thismethod includes the selection of Pfl defective mutants of E. coli andSalmonella typhimurium based on their lack of ability to generate H₂ andCO₂ in the absence of formate, when they are incubated under anaerobiccondition in media containing glucose or pyruvate. However, such aselection method cannot be used for selection of Pfl defective mutantsof lactic acid bacteria, since these organisms lack the enzyme thatcatalyses production of H₂ and CO₂ from formate.

Accordingly, the prior art does not contain any guidance with respect todesigning a feasible method of isolating a lactic acid bacterial Pfldefective (Pfl⁻) mutant.

Experiments performed by the inventor with the minimal medium BA (Clarkand Maaløe, 1967) for E. coli, showed that this medium did not supportthe aerobic growth of lactic acid bacteria. However, if cultivated inthis medium together with E. coli the growth of lactic acid bacteria wassupported, indicating that E. coli produces a factor needed for thegrowth of the lactic acid bacteria. It has later been found that thisgrowth factor is acetate, which led to the development of the DN-medium(Dickely et al., 1995).

It has now surprisingly been found that wild-type strains of lactic acidbacteria such as strains of Lactococcus and Streptococcus including asexamples Lactococcus lactis and Streptococcus thermophilus strains underanaerobic conditions grow well on the DN-medium (Dickely et al., 1995)in the absence of acetate. These unexpected findings have made itpossible to develop a novel and simple method for the isolation of Pfldefective lactic acid bacterial mutants based on the finding that suchmutants, in contrast to the phenotypically Pfl⁺ wild-type strains, areunable to grow under anaerobic conditions on DN-medium in the absence ofacetate.

Additionally, having such a method allowing the selection of Pfldefective lactic acid bacterial mutants at hand has made it possible toprovide further mutated cells which in addition to being Pfl⁻ aremutated in one or more genes involved in the citrate/sugar metabolicpathways such as e.g. the ldh gene coding for lactate dehydrogenase(Ldh) so as to provide a variety of metabolically engineered lactic acidbacteria having highly desirable improved characteristics with respectto metabolite (fermentation end product) production.

The above findings have thus opened up for a novel approach forproviding useful metabolically engineered lactic acid bacterial startercultures which approach is based on relatively simple classical randommutagenesis methods or the selection of spontaneously occurring mutantsand which does not involve in vitro genetic engineering. From apractical technological point of view this is advantageous, since inmost countries the use of genetically engineered food starter culturesis still conditional on approval by regulatory bodies.

SUMMARY OF THE INVENTION

Accordingly, the invention provides in a first aspect a method ofisolating a pyruvate formate-lyase (Pfl) defective lactic acidbacterium, the method comprising the steps of

(i) providing a wild-type lactic acid bacterial strain which underaerobic conditions is not capable of growth in the absence of acetate ina medium not containing lipoic acid, but which is capable of growth issuch medium under anaerobic conditions, and

(ii) selecting from said wild-type strain a mutant which under saidconditions essentially does not grow in the absence of acetate.

In a further aspect, the invention relates to a Pfl defective mutantlactic acid bacterium which is obtainable by the above method andhaving, relative to the wild-type strain from which it is derived, atleast one of the following characteristics:

(i) essentially the same growth rate when cultivated under aerobicconditions in M17 medium,

(ii) a reduced growth rate or a reduced rate of acid production whencultivated under anaerobic conditions in M17 medium or in reconstitutedskim milk (RSM),

(iii) essentially no production of formate under the anaerobicconditions of (ii),

(iv) a reduced production of ethanol or acetate under said aboveanaerobic conditions, and/or

(vi) an increased production of at least one α-acetolactate-derivedmetabolite when cultivated under anaerobic conditions in RSM.

In a still further aspect, there is provided a method of isolating a Pfland lactate dehydrogenase (Ldh) defective lactic acid bacterium which isnot capable of growth under anaerobic conditions in the presence ofacetate, said method comprising

initially selecting a Pfl defective lactic acid bacterium in accordancewith the above method, and

(ii) selecting from said Pfl defective lactic acid bacterium a strainwhich is incapable of growing under anaerobic condition in anacetate-containing medium.

The invention pertains in another aspect to a Pfl and Ldh defectivemutant lactic acid bacterium which is not capable of growing underanaerobic conditions in the presence of acetate, said bacterium beingobtainable by the above method of isolating a Pfl and lactatedehydrogenase (Ldh) defective lactic acid bacterium, and having,relative to a wild-type lactic acid bacterium or its Pfl defectiveparent strain, at least one of the following characteristics:

(i) essentially the same growth yield when cultivated under aerobicconditions in M17 medium,

(ii) a reduced capability of converting lactose to lactate,

(iii) an increased production of α-acetolactate, and/or

(iv) an increased production of an α-acetolactate derived metabolite.

In further aspects, the invention relates to a mutant or variant of theabove Pfl and Ldh defective mutant which mutant or variant is capable ofgrowing anaerobically, to a method of producing a food product,comprising adding to the food product starting materials a culture ofany of the above mentioned lactic acid bacteria and a method ofproducing a lactic acid bacterial metabolite, comprising cultivating anyof the above mentioned lactic acid bacteria under conditions where themetabolite is produced, and isolating the metabolite from the culture.

There is also provided a lactic acid bacterial starter culturecomposition comprising any of the above mentioned lactic acid bacteria.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides in a first aspect a method of isolating apyruvate formate-lyase (Pfl) defective mutant lactic acid bacterium. Asused herein the expression “pyruvate formate-lyase defective” indicatesthat the lactic acid bacterial mutant as compared to the wild-typeparent strain has a reduced Pfl activity or that the Pfl activity isabsent irrespective of the growth conditions, Plf activity beingexpressed herein in terms of formate production. Such a mutant strain isalso referred to herein as a strain having a Pfl⁻ phenotype.

As used herein, the expression “lactic acid bacterium” designates grampositive, microaerophilic or anaerobic bacteria which ferment sugar withthe production of acids including lactic acid as the predominantlyproduced acid, acetic acid, formic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found amongLactococcus species, Streptococcus species, Lactobacillus species,Leuconostoc species, Pediococcus species and Brevibacterium species.Also the strict anaerobes belonging to the genus Bifidobacterium isgenerally included in the group of lactic acid bacteria.

A lactic acid bacterial mutant as defined above can be derived byselecting a spontaneously occurring mutant of a wild-type strain of alactic acid bacterium which has the characteristic that it, when it iscultivated under aerobic conditions in a medium which does not containlipoic acid, has a growth requirement for acetate, but which underanaerobic conditions is capable of growing in such a medium in theabsence of acetate. Alternatively, the mutant of the wild-type lacticacid bacterial strain can be provided by subjecting the strain to amutagenization treatment prior to the selection of a mutant having theabove characteristics of the Pfl defective strain.

It is assumed that these different requirements for acetate under theabove aerobic and anaerobic conditions, respectively is caused by thefacts that under aerobic conditions insufficient amounts of acetyl CoAis formed by the lactic acid bacterium due to at least twocircumstances: (i) in the absence of lipoic acid, an essential co-factorfor the activity of the acetyl CoA generating pyruvate dehydrogenasecomplex (PDC), this enzyme complex does not generate acetyl CoA and (ii)the other major acetyl CoA generating enzyme, pyruvate formate-lyase(Pfl) is inactivated in the presence of oxygen. Therefore, under suchaerobic conditions, the wild-type lactic acid bacterium requires acetateas an alternative source of acetyl CoA. In contrast, under anaerobicconditions, the Pfl is activated and assumingly provides acetyl CoA insufficient amounts for growth of the bacterium. As it is mentionedabove, these observations were the starting point for designing thepresent method of isolating a Pfl defective mutant of a lactic acidbacterium as described herein and the use hereof as an intermediate forproviding further modified strains of lactic acid bacteria.

In accordance with one embodiment of the invention, this method providesin a first step the provision of a wild-type lactic acid bacteriumhaving the above acetate requirement characteristics, followed bysubjecting the bacterium to a mutagenization treatment. In accordancewith the invention, suitable mutagens include conventional chemicalmutagens and UV light. Thus, as examples, a chemical mutagen can beselected from (i) a mutagen that associates with or become incorporatedinto DNA such as a base analogue, e.g. 2-amino-purine or aninterchelating agent such as ICR-191, (ii) a mutagen that react with theDNA including alkylating agents such as nitrosoguanidine orhydroxylamine, or ethane methyl sulphonate (EMS).

Although the lactic acid bacterial mutant can be provided by subjectinga parent strain to a chemical mutagenization treatment followed byselecting a Pfl⁻ mutant, it will be understood that it would also bepossible to provide the mutant by selecting a spontaneously occurringmutant in accordance with the selection procedure as described herein.As an alternative to one presently preferred method of providing themutant by random mutagenesis, it is also possible to provide such amutant by site-directed mutagenesis, e.g. by using appropriatelydesigned PCR techniques or by using a transposable element which isintegratable in lactic acid bacterial replicons.

When a mutagenization step is included, the mutagenized strain is,subsequent to the mutagenization treatment, cultivated under anaerobicconditions in a defined medium not containing lipoic acid in the absenceor presence, respectively of acetate, and a mutant strain, which incontrast to the wild-type parent strain essentially does not grow underthese conditions in the absence of acetate, is selected. It is assumedthat such a mutant strain has a defect in the gene coding for the Pflpolypeptide implying that the production of the enzyme is at leastpartially blocked or that the enzyme is produced in an at leastpartially inactive form. This assumption can be affirmed by testing theselected mutant for lack of production of formate or alternatively, areduced pyruvate formate-lyase activity.

When the mutant is provided as a spontaneously occurring mutant theabove wild-type strain is subjected to the selection step without anypreceding mutagenization treatment. The lactic acid bacterial wild-typeparent strain can be selected from any industrially suitable lactic acidbacterial species, i.e. the strain can be selected from the groupconsisting of a Lactococcus species, a Lactobacillus species, aLeuconostoc species, a Pediococcus species, a Streptococcus species anda Bifidobacterium species. In particular useful embodiments, the lacticacid bacterium is a Lactococcus lactis or a Streptococcus thermophilus.Examples of presently preferred lactic acid bacteria are Lactococcuslactis subspecies lactis and Lactococcus lactis subspecies lactis biovardiacetylactis.

A Pfl defective (Pfl⁻ phenotype) mutant lactic acid bacterium which canbe obtained by the above method has, relative to the wild-type parentstrain one or more phenotypically recognizable characteristicsdistinguishing it from the parent strain. Thus, the Pfl⁻ mutant strainmay have essentially the same growth rate when cultivated under aerobicconditions in M17 medium but a reduced growth rate or a reduced acidproduction when cultivated under anaerobic conditions in conventionalmedia such as the M17 medium or reconstituted skim milk (RSM),essentially no production of formate, a reduced production of ethanol oracetate under said above anaerobic conditions and/or an increasedproduction of at least one α-acetolactate-derived metabolite whencultivated under anaerobic conditions, e.g. in RSM.

Several of these characteristics may be desirable for specific purposes.In the production of a food product it may thus be advantageous that thestrain produces lesser amounts of acids, formate, acetate or ethanol,whereas an enhanced production of α-acetolactate derived aroma orflavour compounds can be highly desirable, in particular in theproduction of dairy products. Such desirable compounds include acetoin,diacetyl and 2,3 butylene glycol. In useful embodiments, the productionof such metabolites such as acetoin is increased by at least 50%, morepreferably by at least 100% and in particular by at least 200%. Besidesbeing useful in the manufacturing of a food product, a mutant strainoverproducing α-acetolactate derived metabolites can also be used,in theproduction of the metabolites as such.

In accordance with the invention, a Pfl defective (Pfl⁻) mutant strainis selected from a Lactococcus species, a Lactobacillus species, aLeuconostoc species, a Pediococcus species, a Streptococcus species anda Bifidobacterium species. In this context, one preferred species isLactococcus lactis including Lactococcus lactis subspecies lactis andLactococcus lactis subspecies lactis biovar diacetylactis, e.g. theLactococcus lactis subspecies lactis strain DN221 which has beendeposited under the accession No. DSM 11034, or a Lactococcus lactisstrain having essentially the characteristics of that strain, or theLactococcus lactis subspecies lactis biovar diacetylactis strain DN227which has been deposited under the accession No. 11040, or a Lactococcuslactis strain having essentially the characteristics of that strain.

It will be understood that the Plf defective lactic acid bacterialmutant can be utilized as a host for the cloning of a pfl gene bycomplementation of the defective gene. Importantly, the Plf⁻ strain canalso be used as a parent strain for isolating mutants having furtheruseful enzymatic defects as it will be described in the following.

Lactate dehydrogenase (Ldh) is, as it can be seen from FIG. 1, anotherenzyme which in lactic acid bacteria contribute to the consumption ofthe pyruvate pool, the activity of the enzyme predominantly resulting inthe production of lactate. It was contemplated that the metabolic fluxtowards α-acetolactate and metabolites derived from this intermediatecould be further increased by providing a mutant strain which inaddition to having a defect in the Pfl activity is defective in Ldh.

Therefore, a strategy for isolating and selecting a lactic acidbacterium which in addition to being Pfl defective is also Ldh defective(Ldh⁻), i.e. having the Pfl⁻ Ldh⁻ phenotypes, was developed based on thefollowing considerations: During anaerobic growth of wild-type lacticacid bacteria the NADH being produced in the glycolysis is converted toNAD⁺ during production of lactate and to some extent during theproduction of ethanol. Accordingly, it was hypothesized that a doublemutant having the Pfl⁻ Ldh⁻ phenotype would be unable to grow underanaerobic conditions, i.e. such a strain would have the additionalphenotype Ang⁻ (inability to grow anaerobically). This hypothesis wasbased on the assumption that such a double mutant would be unable toregenerate NAD⁺ from NADH under anaerobic conditions, since Pfl would beblocked by a mutation (whereas under aerobic conditions, NADH can beconverted to NAD⁺ by NADH oxidase), PDC would be blocked due toinhibition by NADH and Ldh would be blocked by mutation. It was thuscontemplated that a Pfl⁻ Ldh⁻ double mutant could grow under aerobicconditions but not under anaerobic conditions.

Based on the above considerations, a method of isolating a Pfl andlactate dehydrogenase (Ldh) defective lactic acid bacterium which is notcapable of growth under anaerobic conditions in the presence of acetate,i.e. a Pfl⁻ Ldh⁻ Ang⁻ phenotype, was developed. The method comprises asa first step, the selection of a Pfl defective lactic acid bacterium inaccordance with the above method, followed by selecting from this Pfldefective bacterium a strain which is incapable of growing underanaerobic conditions in an acetate-containing medium.

In one presently preferred embodiment this method includes the step ofsubjecting, prior to selection of a strain which is incapable of growingunder anaerobic conditions in an acetate-containing medium, the Pfldefective lactic acid bacterium to a mutagenization treatment andsubsequently selecting a mutant which under said conditions essentiallydoes not grow under said anaerobic conditions.

The above method of isolating the Plf and Ldh defective mutant resultsin a strain having an Ldh specific activity which is reduced relative tothat of its parent (Pfl defective) strain. Preferably, the thus selectedmutant has an Ldh specific activity which is less than 10 units/mgprotein of a cell free extract of the bacterium.

Typically, the thus reduced Ldh specific activity corresponds to at themost 50% activity relative to the wild-type or Pfl⁻ parent strain, suchas at the most 25% or preferably, at the most 10% activity such as atthe most 5% relative to the parent strains. It is particularly preferredthat the mutant strain essentially is devoid of Ldh activity.

The mutagenization step whereby the Pfl⁻ Ldh⁻ Ang⁻ mutant is producedfrom the Pfl⁻ mutant can be performed according to the methods asdescribed above for the mutagenization of the wild-type strain. Itfollows from the above description of this initial step of providing thePfl⁻ mutant that useful strains can be selected from the groupconsisting of a Lactococcus species, a Lactobacillus species, aLeuconostoc species, a Pediococcus species, a Streptococcus species anda Bifidobacterium species. A presently preferred lactic acid bacteriumis Lactococcus lactis including Lactococcus lactis subspecies lactis andLactococcus lactis subspecies lactis biovar diacetylactis.

In accordance with the invention there is also provided a Pfl and Ldhdefective mutant lactic acid bacterium which is obtainable by the abovemethod. In addition to its Pfl⁻ Ldh⁻ Ang⁻ phenotypes, such a mutantstrain can be distinguished from a wild-type lactic acid bacterium orits Pfl defective parent strain in one or more further characteristics.Thus, the mutant strain may have essentially the same growth yield whencultivated under aerobic conditions in M17 medium, a reduced capabilityof converting lactose to lactic acid/lactate, increased production ofα-acetolactate and/or an increased production of an α-acetolactatederived metabolite. Surprisingly, the production of α-acetolactateand/or metabolites derived from α-acetolactate was not only increasedunder aerobic conditions where the mutant strain can grow, but alsounder anaerobic conditions where essentially no growth occurred.

As it is shown in the below Examples, the increase of production ofα-acetolactate and metabolites derived therefrom was of a significantmagnitude. Thus, Pfl⁻ Ldh⁻ Ang⁻ mutants according to the inventionpreferably have a production of α-acetolactate and/or metabolitesderived therefrom which, relative to a wild-type strain of the samespecies, is increased by at least 50%, such as by at least 100%. It iseven more preferred the production is increased by at least 200% such asat least 1000%.

In accordance with the invention, the Pfl⁻ Ldh⁻ Ang⁻ mutant can be ofany lactic acid bacterial species selected from a Lactococcus species, aLactobacillus species, a Leuconostoc species, a Pediococcus species, aStreptococcus species and a Bifidobacterium species. One preferredspecies is Lactococcus lactis including Lactococcus lactis subspecieslactis such as the strain designated DN223 which is described in thefollowing and which is deposited under the accession No. DSM 11036 or aLactococcus lactis strain having essentially the characteristics of thatstrain, and Lactococcus lactis subspecies lactis biovar diacetylactis.

As a result of the enzyme defects of the present Pfl⁻ Ldh⁻ Ang⁻ lacticacid bacterial mutant, such a mutant is capable of converting asubstantial proportion of the intracellular pyruvate pool toα-acetolactate and further to one or more of the metabolites which canbe formed from this intermediate compound, including acetoin, butanedioland/or diacetyl which latter compound can be formed by chemicallyoxidizing α-acetolactate. Thus, in one preferred embodiment, the Pfl⁻Ldh⁻ Ang⁻ mutant is capable of converting at least 15% of pyruvate beingcatabolized to acetoin, more preferably at least 30%. In even morepreferred embodiments, this conversion is at least 40%, such as at least50% or even at least 60%.

In a further aspect, the invention relates to a mutant or variant of theabove Pfl⁻ Ldh⁻ Ang⁻ mutant lactic acid bacterium which is capable ofgrowing anaerobically. Such a mutant or variant strain can be providedby selecting a spontaneous mutant of the above mutant bacterium, whichmutant or variant strain can grow anaerobically. Alternatively, themutant or variant strain can be made by subjecting the Pfl⁻ Ldh⁻ Ang⁻mutant to a further mutagenization treatment in accordance with a methodas described above, and selecting a strain being capable of growinganaerobically. It is contemplated that such mutants or variants wouldhave regained the ability to convert NADH to NAD⁺ under anaerobicconditions, either by mutations in systems secondary to Ldh or Pfl, orby reversion of the Pfl⁻ phenotype to Pfl⁺ phenotype. In wild-typelactic acid bacteria, the level of NADH is high and it can be oxidizedvia lactate and/or ethanol production, i.e. via the pyruvate metabolism.The implication hereof is that lactic acid bacteria produce relativelyhigh levels of lactate and/or ethanol as compared to aerobic conditions.From this it also follows that the metabolites having an aroma effect(diacetyl, acetoin) are only produced at relatively low levels.

It was found that this general picture was still found in the presentAng⁺ mutant or variant of the Pfl⁻ Ldh⁻ Ang⁻ mutant. However, it wassurprisingly found that such a mutant/variant has, relative to itsparent strain and to the wild-type strain, a significantly alteredproduction of aroma compounds under anaerobic growth conditions. Thus,the above Ang⁺ mutant/variant may be one which has a production ofacetaldehyde which relative to the original wild-type strain isincreased at least 2-fold, such as at least 5-fold or even at least8-fold. The mutant variant may also have a production of the diacetylprecursor α-acetolactate which, also relative to the wild-type strain isincreased at least 5-fold such as at least 10-fold.

Also, the production of acetoin and/or formate may be significantlyincreased in such a mutant/variant. Thus, as one typical example, themutant/variant is one which, when grown anaerobically in reconstitutedskim milk powder, produces in excess of 1 mM acetoin and/or in excess of10 mM formate.

A mutant or variant having the latter characteristic is assumingly Ldhdefective but has the wild-type Pfl activity, i.e. it has the phenotypePfl⁺ Ldh⁻ Ang⁺. One example of such a strain is the Lactococcus lactissubspecies lactis DN224 deposited under the accession No. DSM 11037 or aLactococcus lactis strain having essentially the characteristics of thatstrain. Another example of the present mutant or variant is a strainwhich is Pfl defective and has the wild-type Ldh activity, i.e. havingthe phenotype Pfl⁻ Ldh⁺ Ang⁺.

In addition to being a starting material for providing further lacticacid bacterial mutants or variants, the above Pfl⁻ Ldh⁻ Ang⁻ mutant canbe utilized as host for cloning of genes which can restore the abilityof the mutant to grow under anaerobic conditions.

Such a mutant can also, as it is described above by way of example, beused for selecting further mutants having regained the capability ofgrowing anaerobically e.g. due to 30 mutations whereby an increasedamount of one or more NADH oxidoreductases is produced. Suchoxidoreductases include diacetyl reductase (Dr) and Ldh.

The mutant can also be one in which the mutation results inoverproduction and/or enhanced activity of an enzyme, the activity ofwhich can be limiting for a pathway in which a NADH dependentoxidoreductase is involved. Such an overproduction or enhanced activitycan e.g. be of the α-acetolactate synthetase (Als), the increasedproduction or activity of which would in turn result in an increasedproduction of substrate for diacetyl reductase. Alternatively, themutation may result in the above enzyme having an increased activity.

NADH dependent oxidoreductases require a substrate. Thus, as an example,acetoin is the substrate for the oxidoreductase diacetyl reductase (seeFIG. 1). Accordingly, it is contemplated that the above Pfl⁻ Ldh⁻ Ang⁻mutant can be used for selecting a mutant which does not grow, even ifthe oxidoreductase substrate such as acetoin is added to the medium.Such a mutant assumingly will have a defect in one or more of itsoxidoreductases e.g. diacetyl reductase.

Any of the above mutants or variants are potentially useful in theproduction of food products and accordingly, the invention relates in afurther aspect to a method of producing a food product which methodcomprises that a culture of a lactic acid bacterium as described hereinis added to the food product starting materials which are then keptunder conditions appropriate for the bacteria to grow and/or to bemetabolically active. The purpose of the addition of the lactic acidbacteria depends of the food product. In some instances, a lactic acidbacterium according to invention is used to provide an increasedproduction in the food product, such as e.g. a dairy product, of aparticularly desirable aroma compound, such as diacetyl, acetoin oracetaldehyde. Other examples of food products where use of the presentmutant strains is contemplated include meat products, vegetables, bakeryproducts and wine.

It will also be understood that the presently provided strains will behighly useful as production strains in the manufacturing of lactic acidbacterial metabolite compounds including the above aroma compounds.Accordingly, the invention encompasses in a still further aspect amethod of producing a lactic acid bacterial metabolite. Such a methodcomprises cultivating one or more of the lactic acid bacteria asdisclosed herein in a suitable medium under industrially feasibleconditions where the metabolite is produced, and isolating, if required,the metabolite from the culture. The metabolite can be isolated inaccordance with any suitable conventional method of isolating theparticular compound(s) from the cultivation medium. It is also possibleto use the cultivation medium containing the outgrown culture of lacticacid bacteria directly as a source of one or more metabolites.

A specific example of such a production method for a lactic acidbacterial metabolite is a method of producing what is normally referredto in the art as “starter distillate” which is a diacetyl-containingflavouring product conventionally made by cultivating a conventionalwild-type starter culture strain of a lactic acid bacterium whichproduces acetoin and/or diacetyl in a suitable.medium and isolating themetabolites by distillation to provide a concentrate of the metabolites.This product is used for flavouring of butter, margarine, spreads,cereal products and pop-corn. It has been found that by using thestrains DN223 or DN224, such a starter distillate can be obtained thathas a content of diacetyl which, in comparison with a conventionalstarter distillate, is at least 2-fold.

It is convenient to provide the lactic acid bacterium according to theinvention, both when it is used as a food production strain and as aproduction strain for metabolites, as a lactic acid bacterial starterculture composition comprising the lactic acid bacterium selected forthe specific use. Typically, such compositions contain the bacterium inconcentrated form e.g. at a concentration of viable cells (colonyforming units, CFUS) which is in the range of 10⁵ to 10¹³ per g of thecomposition such as a range of 10⁶ to 10¹² per g. Additionally, thestarter culture composition may contain further components such asbacterial nutrients, cryoprotectants or other substances enhancing theviability of the bacterial active ingredient during storage. Thecomposition can be in the form of a frozen or freeze-dried composition.

The invention is further illustrated in the following examples and thedrawings wherein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the pyruvate metabolism in Lactic acid bacteria; theshown enzymatic pathways are: PFL, pyruvate formate-lyase; PDC, pyruvatedehydrogenase complex; LDH, lactate dehydrogenase; ALS, acetolactatesynthetase; ILVB, second acetolactate synthetase; ALD, acetolactatedecarboxylase; DR, diacetyl reductase,

FIGS. 2(a-f) illustrates pH, production of formate (HCOOH), acetate(HAc) and ethanol (EtOH) for Lactococcus lactis subspecies lactisCHCC373 and the mutant DN221 derived therefrom when these strains arecultivated under anaerobic conditions in reconstituted skim milk (RSM),

FIGS. 3(a-f) illustrates OD₆₀₀, production of formate (HFo), acetate(HAc) and ethanol (EtOH) for Lactococcus lactis subspecies lactisCHCC373 and the mutant DN221 derived therefrom when these strains arecultivated under anaerobic conditions in M17 medium, and

FIGS. 4(a-d) shows the growth, acidification and acetoin production ofLactococcus lactis subspecies lactis CHCC373 and mutants or variantshereof (DN221-DN226) as described in the following examples. The strainslisted were grown from single colonies of the respective strainsovernight in 10 ml M17 medium aerobically (+, hatched bars) andanaerobically (−, open bars). The following day, OD₆₀₀. pH and acetoinproduction were measured.

EXAMPLES

Materials and Methods

1. Bacterial strains, media and growth conditions

The following lactic acid bacterial strains were used in the examples:Lactococcus lactis subspecies lactis strains 1FHCY-1, MG1363 and CHCC373(Chr. Hansen Culture Collection), Lactococcus lactis subspecies lactisbiovar diacetylactis DB1341 and Streptococcus thermophilus strainCHCC2134 (Chr. Hansen Culture Collection).

As growth media were used: (i) M17 medium (Terzaghi et al. 1975); (ii)the defined phosphate-buffered DN-medium (Dickely et al. 1995) with orwithout NaAcetate (DN or DN-Ac, respectively). M17 medium was obtainedby adding the following ingredients to 1,000 ml of glass-distilled waterin a 2-liter flask: polypeptone (BBL, Cockeysville, Md.), 5.0 g; Phytonepeptone (BBL), 5.0 g; yeast extract (BBL), 2.5 g; beef extract (BBL),5.0 g; lactose (May and Baker Ltd., Dagenham, England), 5.0 g; ascorbicacid (Sigma Chemical Co., St. Louis, Mo.), 0.5 g; β-sodium GP (grade II,Sigma Chemical Co.), 19.0 g; and 1.0 M MgSO₄.7H₂O (May and Baker, Ltd.),1.0 ml. The DN-medium does not contain lipoic acid, but was supplementedwith NaFormate at a concentration of 0.6%; and (iii) reconstituted skimmilk, RSM containing 9.5% low heat skim milk powder (Milex 240 lh, MDFoods, Denmark).

The strains were cultivated at 30° C. and growth was monitored bymeasuring the optical density (OD) at 600 nm and/or pH. Anaerobicconditions for growth on agar plates were obtained by incubation in asealed container using the Anaerocult® A system (Merck, Darmstadt,Germany). In the following, anaerobic growth conditions for cultures inliquid media means cultivation without shaking and aerobic cultivationmeans growth under shaking.

2. Mutagenesis of L. lactis

A single colony of L. lactis was inoculated in 10 ml DN-medium andincubated for 16 hours under vigorous shaking. To the outgrown culture150 μl of ethyl methane sulphonate (EMS, Sigma) was added and themixture was incubated further under shaking. After 2 hours, 10 tubeseach containing 2 ml DN-medium were each inoculated with 0.2 ml of themutagenized culture. The tubes were incubated until the following dayunder shaking for phenotypic expression. Sterile glycerol was added to afinal concentration of 15% (v/v) and the cultures were stored at −70° C.until use.

3. Determination of Lactate Dehydrogenase Activity

A single colony of L. lactis was inoculated in 10 ml M17 medium andcultivated overnight. After cooling for 15 min. on ice, the cells wereharvested by centrifugation at 7000 rpm for 5 min. at 4° C., washed in 5ml ice-cold Ldh assay buffer (50 mM Tris-Acetate pH 6.0, 0.5 mMFructose-1,6-diphosphate) and resuspended in 1 ml ice-cold Ldh assaybuffer. The resuspended cells were transferred to a 5 ml glass tube andsonicated on ice using a Branson Sonifier 250 at the followingparameters: timer, 4 min.; duty cycle 25%; output 4. Subsequent to thesonication, the content of the tube was transferred to an ice-coldEppendorf tube and centrifuged at 15,000×g for 5 min. at 4° C. Thesupernatant was transferred to a new ice-cold Eppendorf tube. The Ldhspecific activity of the cell-free extract was measured at 25° C. in thefollowing manner: 5 μl of cell-free extract was added to 495 μl Ldhassay buffer containing 0.2 mM NADH and 25 mM pyruvate. As control, anassay without pyruvate was used. The conversion of NADH to NAD⁺ wasfollowed spectrophotometrically over time at 340 nm using a Spectronic®Genesys 5 spectrophotometer. One unit corresponds to the conversion of 1μmol NADH min⁻¹ ml⁻¹ cell-free extract. The specific activity isexpressed in units/mg protein. For measuring the protein concentrationof the cell-free extract, the Bicinchoninic acid (BCA) assay (Pierce,Rockford, U.S.A.) was used with Albumin Standard (Pierce) as proteinstandard.

4. Determination of L. lactis Fermentation End Products

Overnight cultures of the L. lactis strains were inoculated in therespective media and incubated for 24 hours under the relevant growthconditions. Samples were collected and analyzed by HPLC and HS-GC forvarious compounds produced during the cultivation as described byHoulberg (1993, 1995a, 1995b). In certain experiments acetoin wasmeasured as follows: 1 ml culture was transferred to an Eppendorf tubeand centrifuged at 15,000×g, 5 min at 4° C. to remove the cells. Thesupernatant was transferred to a new tube and kept on ice until theacetoin level was measured colometrically using the method of Westerfeld(1945).

Example 1

Acetate Requirement for Growth of L. lactis

Initially, the L. lactis subspecies lactis strains 1FHCY-1 and MG1363were tested for growth on DN-medium with (DN) or without (DN-Ac)acetate, respectively.

The above mentioned strains were streaked onto DN and DN-Ac agar plates,respectively. The plates were incubated for 24 hours under anaerobic andaerobic conditions, respectively. The results are summarized in Table 1below:

TABLE 1 Acetate requirement of 1FHCY-1 and MG1363 Aerobic Anaerobic +Ac−Ac +Ac −AC 1FHCY-1 +++ − +++ +++ MG1363 +++ − +++ +++ +++: colony size0.5-1 mm; −: no growth after prolonged incubation

The tested L. lactis strains have an absolute requirement for acetateunder aerobic growth conditions.

The wild-type strain Lactococcus lactis subspecies lactis CHCC373 wasselected from the culture collection of Chr. Hansen A/S, Hørsholm,Denmark and tested for its growth requirement for acetate under aerobicand anaerobic conditions respectively by streaking a liquid culture ofthe strain onto a series of DN-medium plates containing increasingconcentrations of NaAcetate in the range of from 0 to 0. 2% (w/v).

Under aerobic conditions weak growth was observed at 0.01%; NaAcetateand at 0.02% full growth was observed. No growth was observed atconcentrations below 0.005% NaAcetate. Under anaerobic conditions fullgrowth was observed at 0-0.2% NaAcetate.

In the following experiments, DN-medium with 0.1% NaAcetate (DN) or notcontaining NaAcetate (DN-Ac) was used.

Example 2

Isolation of Pfl Defective Mutants of Lactococcus lactis subspecieslactis CHCC373 and Lactococcus lactis subspecies lactis biovardiacetylactis DB1341 and characterization hereof

2.1. Isolation of Mutants

Mutagenized stocks of the strains CHCC373 and DB1341 were prepared asdescribed above and plated in dilutions onto DN-medium agar plates whichwere incubated aerobically for 24 to 48 hours. From these plates, 980colonies of each strain were selected and streaked onto DN and DN-Acagar plates, respectively and these plates were incubated for 24 hoursunder anaerobic conditions. Two strains designated DN220 and DN221,respectively from the mutagenized CHCC373 strain and one straindesignated DN227 from the mutagenized DB1341 strain which were unable togrow in the absence of acetate under anaerobic conditions were selected.

Chromosomal DNA was isolated from DN220, DN221 and CHCC373, respectivelyand digested with EcoRI, and the fragment patterns were compared usingagarose gel electrophoresis. The fragment patterns showed that bothDN220 and DN221 originated from CHCC373. DN221 was selected for furtherexperiments.

Samples of DN220, DN221 and DN227, respectively were deposited withDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, MascheroderWeg 1b, D-38124 Braunschweig, Germany on Jun. 26, 1996 under therespective accession Nos. DSM 11033, DSM 11034 and DSM 11040.

2.2. Growth of DN221 in M17 Medium and RSM

CHCC373 and DN221 were inoculated in M17 and the cultures were incubatedunder aerobic and anaerobic conditions, respectively. Under aerobicgrowth conditions, DN221 and CHCC373 did grow equally well as judged bythe OD₆₀₀ and the pH. However, the growth rate of DN221 in M17 underanaerobic conditions was considerably lower than that of CHCC373 and itdeclined at a lower cell mass. These results showed that absence ofacetate in M17 was not the reason for the slower growth rate of theselected mutant strain but indicated that an essential characteristicnecessary for anaerobic growth is lacking in DN221 as compared toCHCC373. These results are consistent with the assumption that DN221 hasa defect in its Pfl activity resulting in a requirement for acetate anda lower growth rate under anaerobic conditions as compared to CHCC373.

2.3. Analysis of Fermentates for Various End Products

Single colonies of CHCC373 and DN221, respectively were inoculated inM17 and RSM, respectively and these cultures were incubated for 24 hoursunder anaerobic conditions. Samples were collected taken analyzed forcontent of fermentation end product compounds according to the abovemethods.

The results which are summarized in FIGS. 2 and 3 show that formate isnot produced by DN221, but is produced by CHCC373 at a high level. Thisconfirms that DN221 lacks Pfl activity This is further confirmed by thelow levels of ethanol and acetate produced by DN221 as compared to itsparent strain, CHCC373.

Example 3

Isolation of Pfl and Ldh Defective Mutants and Characterization Hereof

3.1. Isolation of Mutants

A stock of DN221 was mutagenized as described above under Materials andMethods, and the mutagenized cells were plated in dilutions ontoDN-medium agar plates which were incubated aerobically for 24-48 hours.From theses plates, 980 colonies were selected and each colony wasstreaked onto two DN plates and incubated 24 hours under anaerobic andaerobic conditions, respectively. Two strains (DN222 and DN223) whichwere unable to grow under anaerobic conditions were selected.

Chromosomal DNA was isolated from DN222, DN223 and CHCC373, respectivelyand digested with EcoRI. The fragment patterns were compared usingagarose gel electrophoresis. The fragment patterns showed that bothDN222 and DN223 originate from CHCC373.

Samples of DN222 and DN223, respectively were deposited with DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b,D-38124 Braunschweig, Germany on Jun. 26, 1996 under the respectiveaccession Nos. DSM 11035 and DSM 11036.

3.2. Testing for Lactate Dehydrogenase (Ldh) Activity

The Ldh activity of DN221, DN222, DN223 and CHCC373 was analyzed inaccordance with the method described above, and the results are shown inTable 3.1 below.

TABLE 3.1 Ldh activity of DN221, DN222, DN223 and CHCC373 Strain CHCC373DN221 DN222 DN223 Spec. activity^(a) 0.30 0.28 0.12 0.59 NADH oxidaseSpec. activity^(b) 21.40 19.10 16.00 0.70 Ldh % Ldh act. 100.00 89.0075.00 3.00 ^(a)units/mg protein; units, μmol NAD+ min⁻¹ ml⁻¹ extracts.Assay without pyruvate. ^(b)as ^(a) but assay with pyruvate.

These results show that DN223 has a defect in the Ldh activity havingonly 3% of the activity of CHCC373, whereas DN222 has a Ldh activitysimilar to that of CHCC373. Thus it can be concluded that DN223 is Pfland Ldh defective.

3.2. Growth in M17 and Formation of End Products Under AerobicConditions

Single colonies of CHCC373, DN221, DN222 and DN223, respectively wereinoculated in M17 and incubated under aerobic conditions. The results ofmeasurements of OD₆₀₀ and pH for the outgrown cultures are shown intable 3.2 below.

TABLE 3.2 OD₆₀₀ and pH of CHCC373, DN221, DN222 and DN223 CHCC373 DN221DN222 DN223 OD^(a) 3.26 ± 0.16 3.16 ± 0.02  1.7 ± 0.08 3.22 ± 0.16pH^(b)  5.7 ± 0.06 5.71 ± 0.08 5.84 ± 0.16 6.21 ± 0.08 ^(a)a singlecolony was inoculated in 10 ml M17 medium and cultivated for 24 hoursfollowed by measuring the OD₆₀₀. ^(b)as ^(a) except that the pH wasmeasured.

Under these conditions DN221, DN223 and CHCC373 had similar growthyields as judged by the OD₆₀₀ measurements. However, the OD₆₀₀ of DN222was only about half the OD₆₀₀ of the wild-type strain. DN221 and DN222both acidified the medium to the same pH level, whereas DN223 onlyacidified the medium slightly, even though the growth as judged by theOD₆₀₀ was similar to that of CHCC373, confirming that DN223 is Ldhdefective.

Overnight cultures from single colonies of CHCC373, DN221, DN222 andDN223 were inoculated in M17 and RSM were incubated for 24 hours underaerobic conditions and samples were taken for analysis of end productsas described above. Results from the analysis are shown in Table 3.3below.

TABLE 3.3 End product formation in M17 AA EtOH DAc HMEK ALA HAc LactoHLac mM* mM* mM* mM* mM* mM* mM* mM* M17 0.02 0.05 0.00 0.2 0.00 1.7 5.50.00 CHCC373 0.15 0.06 0.02 2.2 0.03 23.3 0.00 24.2 DN221 0.15 0.08 0.022.7 0.03 25.0 0.00 24.2 DN222 0.12 0.03 0.03 1.6 0.02 16.7 0.00 22.0DN223 0.12 0.32 0.07 14.2 0.15 18.3 0.00 4.4 *see abbreviation belowAbbreviations: AA acetaldehyde ALA acetolactate DAc diacetyl EtOHethanol HAc acetic acid HLac lactic acid HMEK acetoin Lacto lactose

CHCC373 produces almost equal amounts of acetate and lactate. Underaerobic conditions DN221 produce similar amounts of end products as doesCHCC373. DN222 produces less acetate and equal amounts of lactate asdoes CHCC373. The defect in DN222 is unknown. DN223 produces very smallamounts of lactate as compared to CHCC373. DN223 converted the majorpart of pyruvate to acetoin instead of lactate. This change in pyruvatecatabolism is also reflected in that the aroma compound diacetyl wasincreased 3-4 fold as compared to CHCC373 and in that about 55% of thecatabolized pyruvate passed via α-acetolactate (ALA) to acetoin (HMEK).The percentage is probably higher as the butanediol production was notmeasured.

Example 4

Isolation and Characterization of Spontaneous Mutants of DN223

4.1. Isolation of Mutants

A liquid culture was made from a single colony of DN223 and incubatedunder aerobic conditions overnight. Approximately 10⁸ cells weretransferred to DN-medium agar plates which were incubated underanaerobic conditions. Three strains designated DN224, DN225 and DN226were isolated based on their ability to grow under anaerobic conditions.The three strains are all mutants or variants of DN223 having regainedthe ability to convert NADH to NAD⁺ under anaerobic conditions either bymutations in secondary systems to Ldh and Pfl or by reversion of the Pflor the Ldh defect.

Chromosomal DNA was isolated from DN224, DN225, DN226 and CHCC373,respectively and digested with EcoRI. The fragment patterns werecompared using agarose gel electrophoresis. The fragment patterns showedthat DN224, DN225 and DN226 all originate from CHCC373.

Samples of DN224, DN225 and DN226, respectively were deposited withDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, MascheroderWeg 1b, D-38124 Braunschweig, Germany on Jun. 26, 1996 under therespective accession Nos. DSM 11037, DSM 11038 and DSM 11039.

4.2. Growth in M17 and Acetoin Formation

Single colonies of CHCC373, DN221, DN222, DN223, DN224 and DN226,respectively were inoculated in M17 and incubated overnight underanaerobic and aerobic conditions, respectively. The final OD₆₀₀ and pHwere measured and samples were collected and analyzed for content ofacetoin. The results are shown in FIG. 4.

All of the tested strains, except DN222, grew well under aerobicconditions. Under anaerobic conditions, DN221, DN222 and DN223 hadsevere growth defects, DN223 being the most growth inhibited strain.Measurements of the pH reflected the growth pattern of the strainsexcept for DN223, DN224 and DN226 grown under aerobic conditions. Theseresults showed that DN224 and DN226 have reduced acidifying capacitycompared to CHCC373, possibly caused by a defect in Ldh. Under bothaerobic and anaerobic conditions DN225 has a growth yield equal toCHCC373 indicating that this strain had lost the Ldh defect.

Under aerobic conditions, the strains DN223, DN224 and DN226 producedacetoin in the range of 1100-1200 ppm which is 4-6 times more than theother strains (about 200 ppm). Under anaerobic conditions, DN223produced about 10-fold more acetoin than the strains DN222 and CHCC373,even though almost no growth was observed as judged by the OD₆₀₀ value.DN224 and DN226 produced more than 10 ppm acetoin under anaerobicconditions, which is considerably more than the 2.5 ppm produced byCHCC373 and DN222. The high concentrations of acetoin produced by thethree strains indicates that these strains have the potential ofproducing high amounts of diacetyl.

Among the above spontaneous mutants, DN224 was selected for furtherstudies of fermentation end product formation.

4.3. Growth in RSM and Formation Herein of End Products

Single colonies of CHCC373, DN221, DN223 and DN224 were inoculated in 10ml M17 and incubated overnight. The final OD₆₀₀ and pH were measured.The results are shown in Table 4.1 below.

TABLE 4.1 Growth (OD₆₀₀) of CHCC373, DN221, DN223 and DN224 in M17 OD₆₀₀pH CHCC373 3.34 5.7 DN221 3.22 5.72 DN223 3.44 6.29 DN224 3.28 6.29

Subsequently 2×200 μl of each culture was transferred to 2×10 ml RSM andincubated over night under aerobic and anaerobic conditions,respectively. pH was measured and results are shown in Table 4.2 below.

TABLE 4.2 Growth (pH) of CHCC373, DN221, DN223 and DN224 in RSM pHAnaerobic Aerobic milk 6.82 6.8 CHCC373 4.37 5.01 DN221 5.02 5.23 DN2236.56 6.08 DN224 5.13 6.06

The growth in RSM under aerobic conditions, as judged by pH, appears tobe as in M17 indicating, that the acidifying capacity is independent ofthe media used.

From all cultures samples were taken for analysis of end products. Theresults are shown in Table 4.3 below.

TABLE 4.3 End product formation in RSM HCit* HAc* Lacto* HLac* AA* EtOH*DAc* HMEK* ALA* MeFo* mM mM mM mM mM mM mM mM mM mM RSM 9.5 0 132 1. 0.10 0 0 0.1 0.2 RSM 9.5 0 131 1.1 0.01 0 0 0.16 0.01 0.2 CHCC373 aerobic9.5 5 109 46.7 0.08 0.06 0.13 10.93 0.24 0.13 CHCC373 aerobic 9.5 5 10847.8 0.08 0.07 0.14 11.47 0.21 0.11 CHCC373 anaerobic 8.1 1.7 96 70 0.020.93 0.14 0 1.09 CHCC373 anaerobic 9.5 1.7 106 76.7 0.02 0.88 0 0 0 1.35DN221 aerobic 9.5 3.3 117 38.9 0.05 0 0.02 1.11 0.02 0.01 DN221 aerobic9.5 3.3 117 38.9 0.05 0.02 0.02 1.07 0.02 0.01 DN221 anaerobic 9 1.7 11451.1 0.05 0.36 0.01 0.39 0.01 0.11 DN221 anaerobic 8.6 1.7 109 46.7 0.050.35 0.01 0.22 0.01 0.11 DN223 aerobic 9 119 6.6 0.04 0.05 0.12 10.540.21 0.13 DN223 aerobic 10 5 120 6.7 0.05 0.05 0.13 10.73 0.2 0.13 DN223anaerobic 10 1.7 131 3.3 0.01 0.06 0.01 0.3 0.01 0.28 DN223 anaerobic1.7 122 3.3 0.01 0.05 0.01 0.36 0.01 0.26 DN224 aerobic 10 5 123 7.80.05 0.04 0.13 10.84 0.21 0.15 DN224 aerobic 9.5 5 119 6.7 0.05 0.040.13 10.42 0.21 0.13 DN224 anaerobic 9 6.7 115 15.6 0.14 13.19 0.01 4.140.09 12.52 DN224 anaerobic 2 6.7 119 16.7 0.16 13.83 0.01 3.68 0.0813.35 *see abbreviations below Abbreviations: AA acetaldehyde ALAacetolactate DAc diacetyl EtOH ethanol HAc acetic acid HCit citrate HLaclactic acid HMEK acetoin Lacto Lactose MeFo formate

None of the strains fermented citrate as would be expected of a L.lactis subspecies lactis. The wild-type strain CHCC373 grown underaerobic conditions produced relatively high amounts of acetoin,diacetyl, α-acetolactate, acetaldehyde and acetate, but relatively lowamounts of ethanol and lactate as compared to the production hereofunder anaerobic conditions.

From the results obtained from DN224, it can be seen that the levels ofthe different aroma compounds have changed significantly duringanaerobic growth. The level of acetaldehyde was increased about 8-fold,the diacetyl precursor α-acetolactate had increased more than 10-fold ascompared to the level hereof of CHCC373.

However, it is assumed that the potential for diacetyl production ismuch higher, as the amount of acetoin produced by DN224 compared to theamount of acetoin produced by CHCC373 is significantly higher. Theincrease of formate, ethanol and acetate production and the reduction oflactate production indicates that DN224 has lost the defect in Pfl butis still Ldh defective. This is further verified by the fact that DN224grows under anaerobic conditions which the Pfl and Ldh defective strainDN223 does not.

Example 5

Detection of Acetate Requirement for Growth of Streptococcusthermophilus

Single colonies of Streptococcus thermophilus CHCC2134 was streaken ontoplates of DN agar containing lactose (5 g/L), Na-formiate (20 mg/L) andwith and without acetate. The plates were incubated for 48 hours at 37°C. under aerobic and anaerobic conditions, respectively. Growth occurredas summarized in Table 5.1 below:

TABLE 5.1 Acetate requirement of Streptococcus thermophilus CHCC2134Aerobic Anaerobic With acetate ++ +++ Without acetate − +++ ++: colonysize 0.1-0.5 mm; +++: colony size 0.5-2 mm; −: no growth after prolongedincubation

Since acetate is required for growth at aerobic conditions, the basisexists for the isolation of a mutant strain of Streptococcusthermophilus that has a requirement for acetate under anaerobicconditions, i.e. a Pfl⁻ mutant of that species. Such a mutant straincould, in analogy with the above, be used as the starting material inthe isolation of a second mutant strain being incapable of growing underanaerobic conditions, i.e. a Pfl⁻/Ldh⁻ mutant.

References

1. Dickely F, Nilsson D, Hansen E B, Johansen E. 1995. Isolation ofLactococcus lactis nonsense suppressors and construction of a food-gradecloning vector. Molec. Microbiol., 15, 839-847.

2. Gasson M J, Benson K, Swindell S, Griffin H. 1996. Metabolicengineering of the Lactococcus lactis diacetyl pathway. Lait, 76, 33-40.

3. Houlberg U. 1993. HPLC analysis: Determination of acids &carbohydrates in liquid fermentation media using internal standard.Analytical Procedure 1009, Chr. Hansen A/S.

4. Houlberg U. 1995a. HSGC-In situ derivatization of acids infermentates for physiological investigations. Technical Report 785, Chr.Hansen A/S.

5. Houlberg U. 1995b. HSGC-Determination of volatile organic compoundsand α-acetolactic acid.

6. Hugenholtz J. 1993. Citrate metabolism in lactic acid bacteria. FEMSMicrobiology Reviews, 12, 165-178.

7. Knappe J. 1987. Anaerobic dissimilation of pyruvate. In F. C.Neidhardt (ed.) Escherichia coli and Salmonella typhimurium. Cellularand Molecular Biology. pp 151-155.

8. Platteeuw C, Hugenholtz J, Starrenburg M, van Alen-Boer-rigter I, DeVos WM. 1995. Metabolic engineering of Lactococcus lactis: Influence ofthe overproduction of α-acetolactate synthetase in strains deficient inlactate dehydrogenase as a function of culture conditions. Appl.Environ. Microbiol., 61, 3967-3971.

9. Snoep J L. 1992. Regulation of pyruvate catabolism in Enterococcusfaecalis. Ph. D. thesis, University of Amsterdam, Netherlands.

10. Terzaghi B E, Sandine W E. 1975. Improved medium for the lacticstreptococci and their bacteriophages. Appl. Microbiol., 29, 807-813.

11. Westerfeld W W. 1945. A calorimetric determination of blood acetoin.J. Biol. Chem., 16, 495-502

What is claimed is:
 1. A method of isolating a pyruvate formate-lyase(Pfl) and lactate dehydrogenase (Ldh) defective Lactococcus lactislactic acid bacterium which is not capable of growth under anaerobicconditions in the presence of acetate, said method comprising (i)selecting a Pfl defective Lactococcus lactis lactic acid bacterium, and(ii) selecting from said Pfl defective lactic acid bacterium a strainwhich is incapable of growing under anaerobic conditions in anacetate-containing medium, wherein the strain of Lactococcus lactisselected in (ii) is the Pfl and Ldh defective strain of wherein at least15% of pyruvate being catabolized by the Pfl and Ldh defective strain ofLactococcus lactis lactic acid bacterium selected in step (ii) isconverted to acetoin.
 2. The method of claim 1 where, in step (i) ofclaim 1, the pyruvate formate-lyase (Pfl) defective lactic acidbacterium is selected by (a) providing a wild-type Lactococcus lactislactic acid bacterial strain which under aerobic conditions does notgrow in an acetate-free, lipoic acid-free medium, but which grows insuch medium under anaerobic conditions, (b) subjecting said wild-typestrain to a mutagenization treatment, (c) culturing the thus-treatedstrain, and (d) selecting from said culture a mutant which is a pyruvateformate-lyase (Pfl) defective lactic acid bacterium which underanaerobic conditions does not grow in the absence of acetate.
 3. Amethod according to claim 1 where in step (ii) the Pfl defective lacticacid bacterium is subjected to a mutagenization treatment.
 4. A methodaccording to claim 1 wherein the selected lactic acid bacterium has anLdh specific activity which is at the most 50% of the Ldh specificactivity of the Ldh positive wild type strain from which it is derived.5. A method according to claim 1 wherein the lactic acid bacterium is ofsubspecies lactis.
 6. A culture comprising a Pfl and Ldh defectivemutant Lactococcus lactis lactic acid bacterium which is non-naturallyoccurring, which has been at least partially biologically purified, andwhich bacterium is not capable of growing under anaerobic conditions inthe presence of acetate, said bacterium being obtained by the method ofclaim 1 and having, relative to a wild-type Lactococcus lactis lacticacid bacterium or its Pfl defective parent strain, at least one of thefollowing characteristics: (i) essentially the same growth yield, asmeasured by the OD₆₀₀, when cultivated under aerobic conditions in M17medium, (ii) a reduced capability of converting lactose to lactate,(iii) an increased production of α-acetolactate, and/or (iv) anincreased production of an α-acetolactate derived metabolite selectedfrom the group consisting of acetoin, 2,3-butanediol and diacetyl. 7.According to claim 6 said bacterium having an α-acetolactate productionwhich is increased by at least 100%.
 8. A culture according to claim 6said bacterium having an α-acetolactate derived metabolite productionwhich is increased by at least 100%.
 9. A culture according to claim 6which bacterium is Lactococcus lactis subspecies lactis.
 10. A cultureaccording to claim 6 which bacterium is Lactococcus lactis subspecieslactis DN223 deposited under the accession No. DSM11036 or a strainhaving all of the identifying characteristics as DSM
 11036. 11. Abiologically pure culture of a non-recombinant Pfl and Ldh defectivemutant Lactococcus lactis lactic acid bacterium which is non-naturallyoccuring and which is not capable of growing under anaerobic conditionsin the presence of acetate.
 12. The culture of claim 6 which is a lacticacid bacterial starter culture for use in a food or feed product. 13.The method of claim 2 or 3 in which the mutagenizing treatment compriseexposure to a chemical mutagen.
 14. The method of claim 13 in which thechemical mutagen is ethyl methane sulfonate.
 15. A starter culturecomposition comprising the biologically pure culture according to claim11.
 16. The starter culture composition according to claim 15 furthercomprising cells of other lactic acid bacterial strains.
 17. A methodaccording to claim 12, wherein the selected lactic acid bacterium has anLdh specific activity which is less than 10 units/mg protein of acell-free extract of the bacterium.
 18. The method of claim 1 wherein atleast 30% of pyruvate being catabolized by the lactic acid bacteriumselected in step (ii) is converted to acetoin.
 19. The method of claim 1wherein at least 40% of pyruvate being catabolized by the lactic acidbacterium selected in step (ii) is converted to acetoin.
 20. The methodof claim 1 wherein at least 50% of pyruvate being catabolized by thelactic acid bacterium selected in step (ii) is converted to acetoin. 21.The method of claim 1 wherein at least 60% of pyruvate being catabolizedby the lactic acid bacterium selected in step (ii) is converted toacetoin.